A region of the interleukin-2 (IL-2) promoter known as the RE/AP element is activated in concert by signals that originate from the T cell antigen receptor and the CD28 coreceptor. We show here that the serine-threonine kinase Akt can provide a costimulatory signal for RE/AP activation that is indistinguishable from the signal provided by CD28. This includes the ability of Akt, like antibodies to CD28, to synergize with protein kinase C theta (PKC-theta) in the induction of RE/AP. Retrovirus-mediated expression of activated Akt in primary T cells from CD28-deficient mice is capable of selectively restoring production of IL-2 and interferon gamma, but not IL-4 or IL-5. Our results provide evidence that CD28 costimulation of different cytokines is mediated by discrete signaling pathways, one of which includes Akt.

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The chemokine receptors CCR2 and CCR5 and their respective ligands regulate leukocyte chemotaxis and activation. To determine the role of these chemokine receptors in the regulation of the intestinal immune response, we induced colitis in CCR2- and CCR5-deficient mice by continuous oral administration of dextran sodium sulfate (DSS). Both CCR2- and CCR5-deficient mice were susceptible to DSS-induced intestinal inflammation. The lack of CCR2 or CCR5 did not reduce the DSS-induced migration of macrophages into the colonic lamina propria. However, both CCR5-deficient mice and, to a lesser degree, CCR2-deficient mice were protected from DSS-induced intestinal adhesions and mucosal ulcerations. CCR5-deficient mice were characterized by a greater relative infiltration of CD4+ and NK1.1+ lymphocyte in the colonic lamina propria when compared to wild-type and CCR2-deficient mice. In CCR5-deficient mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN-γ was decreased, corresponding to a Th2 pattern of T cell activation. In CCR2-deficient mice, the infiltration of Th2-type T cells in the lamina propria was absent, but increased levels of IL-10 and decreased levels of IFN-γ may have down regulated mucosal inflammation. Our data indicate that CCR5 may be critical for the promotion of intestinal Th1-type immune responses in mice.

CD28 costimulation is essential for CD4(+) T cell proliferation, survival, interleukin 2 (IL-2) production and T helper type 2 development. To define the nature of the signals that may drive different T cell responses, we have done a structure-function analysis of the CD28 cytoplasmic tail in primary T cells. CD28-mediated T cell proliferation and IL-2 production did not require a particular cytoplasmic domain. In contrast, IL-4 production was driven by the cooperative activity of specific motifs within the CD28 cytoplasmic tail. Using a gene-complementation approach, we provide evidence that one component of this T helper type 2 differentiation signal was mediated by 3-phosphoinositide-dependent protein kinase 1. Thus, different mechanisms underlie the induction of distinct T cell functional responses by CD28.

Lymphocytes deficient in the T cell costimulatory molecule CD28 exhibit defects in cell survival, clonal expansion, and differentiation into effector cells. It is known that CD28-mediated signaling results in the upregulation of the Bcl family member Bcl-XL. To investigate the role that Bcl-XL plays in the various functions of CD28, we expressed Bcl-XL in CD28-deficient primary T lymphocytes using retrovirus-mediated gene transfer. T cells were activated in vitro and infected with Bcl-XL or control retroviruses; this method allows gene expression in activated, cycling cells. Expression of Bcl-XL in naive T cells was achieved by reconstitution of the immune system of lethally irradiated recipient mice with retrovirus-infected purified bone marrow stem cells from CD28−/− or wild-type donor mice. Our studies demonstrate that Bcl-XL prolongs the survival of CD28−/− T cells but does not restore normal proliferation or effector cell development. These results indicate that the various functions of CD28 can be dissociated, and provide an experimental approach for testing the roles of downstream signals in the functions of cellular receptors such as CD28.